Pyrrolo[2,3-D]pyrimidine compounds

ABSTRACT

Described herein is pyrrolo{2,3-d}pyrimidine compounds, their use as Janus Kinase (JAK) inhibitors, pharmaceutical compositions containing this compounds, and methods for the preparation of these compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/619,195 filed Feb. 11, 2015, now allowed; which is a divisional ofU.S. patent application Ser. No. 13/360,963 filed Jan. 30, 2012, nowgranted as U.S. Pat. No. 8,987,283; which is a divisional of U.S. patentapplication Ser. No. 12/542,451 filed Aug. 17, 2009, now granted as U.S.Pat. No. 8,133,899, which claims the benefit of priority to U.S.Provisional Application No. 61/090,371 filed Aug. 20, 2008.

FIELD OF THE INVENTION

Described herein isN-methyl(4-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclohexyl)methanesulfonamide,its analogues, their use as Janus Kinase (JAK) inhibitors,pharmaceutical compositions containing these compounds, and methods forthe preparation of these compound.

BACKGROUND OF THE INVENTION

Protein kinases are families of enzymes that catalyze thephosphorylation of specific residues in proteins, broadly classifiedinto tyrosine and serine/threonine kinases. Inappropriate kinaseactivity, arising from mutation, over-expression, or inappropriateregulation, dys-regulation or de-regulation, as well as over- orunder-production of growth factors or cytokines has been implicated inmany diseases, including but not limited to cancer, cardiovasculardiseases, allergies, asthma and other respiratory diseases, autoimmunediseases, inflammatory diseases, bone diseases, metabolic disorders, andneurological and neurodegenerative disorders such as Alzheimer'sdisease. Inappropriate kinase activity triggers a variety of biologicalcellular responses relating to cell growth, cell differentiation,survival, apoptosis, mitogenesis, cell cycle control, and cell mobilityimplicated in the aforementioned and related diseases.

Thus, protein kinases have emerged as an important class of enzymes astargets for therapeutic intervention. In particular, the JAK family ofcellular protein tyrosine kinases (JAK-1, JAK-2, JAK-3, and Tyk-2) playa central role in cytokine signaling (Kisseleva et al, Gene, 2002, 285,1; Yamaoka et al. Genome Biology 2004, 5, 253)). Upon binding to theirreceptors, cytokines activate JAK which then phosphorylate the cytokinereceptor, thereby creating docking sites for signaling molecules,notably, members of the signal transducer and activator of transcription(STAT) family that ultimately lead to gene expression. Numerouscytokines are known to activate the JAK family.

Accordingly, there remains a need for alternative compounds thateffectively inhibit JAK enzymes, including JAK-1, JAK-2, JAK-3, and/orTyk-2.

SUMMARY OF THE INVENTION

The present invention provides a compound of formula I:

or a pharmaceutically acceptable salt thereof wherein R¹ is —C₁₋₄alky,optionally substituted with hydroxy.

Specifically, a compound of formula I wherein R¹ is methyl.

Specifically, a compound of formula I wherein R¹ is ethyl or cyclobutyl.

In another aspect, the present invention also provides:

pharmaceutical compositions which comprise a pharmaceutically acceptablecarrier and a compound of formula I,

methods for controlling or treating a disorder or condition selectedfrom organ transplant rejections, lupus, multiple sclerosis, rheumatoidarthritis, psoriasis, cancer, osteoarthritis, and diabetes byadministering to a mammal in need a therapeutically effective amount ofa compound of formula I or a pharmaceutically acceptable salt thereof,

methods for controlling or treating a disorder or condition selectedfrom diabetes, cancer, autoimmune thyroid disorders, ulcerative colitis,Crohn's disease, dry eyes, Alzheimer's disease, leukemia, and otherindications where immunosuppression or immunomodulation would bedesirable by administering to a mammal in need a therapeuticallyeffective amount of a compound of formula I or a pharmaceuticallyacceptable salt thereof,

methods for controlling or treating a disorder or condition selectedfrom allergic reaction including allergic dermatitis, eczema, atopicdermatitis, pruritus and other pruritic conditions and inflammatorydisease such as bowel disease in mammal by administering to a mammal inneed a therapeutically effective amount of a compound of formula I or apharmaceutically acceptable salt thereof,

methods for controlling or treating a disorder or condition selectedfrom Asthma and other obstructive airways diseases, including chronic orinveterate asthma, late asthma, airway hyper-responsiveness, bronchitis,bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma,dust asthma, recurrent airway obstruction, and chronic obstructionpulmonary disease by administering to a mammal in need a therapeuticallyeffective amount of a compound of formula I or a pharmaceuticallyacceptable salt thereof,

methods for the inhibition of protein tyrosine kinases or JAK-1, JAK-2,JAK-3 and/or Tyk-2 by administering to a mammal in need of atherapeutically effective amount of a compound of formula I or apharmaceutically acceptable salt thereof,

methods for the inhibition of protein tyrosine kinases or JAK-1, JAK-2,JAK-3 and/or Tyk-2 by administering to a mammal in need of atherapeutically effective amount of a compound of formula I or apharmaceutically acceptable salt thereof, and

methods for the preparation of compounds of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a characteristic X-ray powder diffraction pattern ofN-methyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}methanesulfonamidemaleic acid salt (Form A).

FIG. 2 illustrates Day 27 VAS Scores for Example 1b in flea allergicdogs in the Reducing Flea-associated Pruritus and Dermatitis Assay

FIG. 3 illustrates Seconds of Pruritus per 4 hour recording for example1b in flea allergic dogs in the Reducing Flea-associated Pruritus andDermatitis Assay.

DETAILED DESCRIPTION

With respect to the above compound, and throughout the application andclaims, the following terms have the meanings defined below.

The term “mammal” refers to human or animals including livestock andcompanion animals. The phrase “companion animal” or “companion animals”refers to animals kept as pets. Examples of companion animals includecats, dogs, and horses. The term “livestock” refers to animals reared orraised in an agricultural setting to make products such as food orfiber, or for its labor. In some embodiments, livestock are suitable forconsumption by mammals, for example humans. Examples of livestockanimals include mammals, such as cattle, goats, horses, pigs, sheep,including lambs, and rabbits, as well as birds, such as chickens, ducksand turkeys.

The term “controlling”, “treating” or “treatment” of a disease includes:(1) preventing the disease, i.e. causing the clinical symptoms or signsof the disease not to develop in a mammal that may be exposed to orpredisposed to the disease but does not yet experience or displaysymptoms/signs of the disease; (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its clinicalsymptoms/signs; or (3) relieving the disease, i.e., causing regressionof the disease or its clinical symptoms/signs.

The term “therapeutically effective amount” means the amount of acompound that, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

The term “approximately” if used in defining a peak in an X-ray powderdiffraction pattern is defined as the stated 2-theta value±0.2 degree2-theta. Any determination of whether a crystalline form is the Form Apolymorph and encompassed by the claims should be interpreted in lightof the variability in this test.

“Pharmaceutically acceptable” means suitable for use in mammals,companion animals or livestock animals.

The carbon atom content of various hydrocarbon-containing moieties isindicated by a prefix designating the minimum and maximum number ofcarbon atoms in the moiety, i.e., the prefix C_(i-j) indicates a moietyof the integer “i” to the integer “j” carbon atoms, inclusive. Thus, forexample, C₁₋₄ alkyl refers to alkyl of one to four carbon atoms,inclusive.

The term alkyl refers to straight, branched and a cyclic saturatedmonovalent hydrocarbon groups, but reference to an individual radicalsuch as “propyl” embraces only the straight chain radical, a branchedchain isomer such as “isopropyl” or a cyclic isomer such ascyclopropylmethyl or cyclopentyl being specifically referred to.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers”. Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers”. It will be appreciatedby those skilled in the art that the compound of formula I can exist ascis- and trans-achiral diastereomers. Specifically, the presentinvention provides a compound of formula IA, which has the chemical nameN-methyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}methanesulfonamide,

or a pharmaceutically acceptable salt thereof.

Included within the scope of the described compounds are all isomers(e.g. cis-, trans-, or diastereomers) of the compounds described hereinalone as well as any mixtures. All of these forms, includingenantiomers, diastereomers, cis, trans, syn, anti, solvates (includinghydrates), tautomers, and mixtures thereof, are included in thedescribed compounds.

Stereoisomeric mixtures, e.g. mixtures of diastereomers, can beseparated into their corresponding isomers in a known manner by means ofsuitable separation methods. Diastereomeric mixtures for example may beseparated into their individual diastereomers by means of fractionatedcrystallization, chromatography, solvent distribution, and similarprocedures. This separation may take place either at the level of one ofthe starting compounds or in a compound of formula I itself. Enantiomersmay be separated through the formation of diastereomeric salts, forexample by salt formation with an enantiomer-pure chiral acid, or bymeans of chromatography, for example by HPLC, using chromatographicsubstrates with chiral ligands.

Routes of Administration

In therapeutic use for treating disorders in a mammal (i.e. human andanimals), a compound of the present invention or its pharmaceuticalcompositions can be administered orally, parenterally, topically,rectally, transmucosally, or intestinally. Parenteral administrationsinclude indirect injections to generate a systemic effect or directinjections to the afflicted area. Topical administrations include thetreatment of skin or organs readily accessibly by local application, forexample, eyes or ears. It also includes transdermal delivery to generatea systemic effect. The rectal administration includes the form ofsuppositories. The preferred routes of administration are oral andparenteral.

Pharmaceutical Salts

The compound of formula I may be used in its native form or as a salt.In cases where forming a stable nontoxic acid or base salt is desired,administration of the compound as a pharmaceutically acceptable salt maybe appropriate. Pharmaceutically acceptable salts of the compounds offormula I include the acetate, ascorbate, aspartate, benzoate, besylate,bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate,edisylate, etoglutarate, esylate, formate, fumarate, gluceptate,gluconate, glucuronate, glycerophosphate, hexafluorophosphate,hibenzate, hydrochloride/chloride, hydrobromide/bromide,hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate,mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate,orotate, oxalate, palmitate, pamoate, phosphate/hydrogenphosphate/dihydrogen phosphate, saccharate, stearate, succinate,tartrate, tosylate and trifluoroacetate salts.

Composition/Formulation

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulation, dragee-making, levigating, emulsifying,encapsulating, entrapping, lyophilizing processes or spray drying.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morepharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compound intopreparations, which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Pharmaceuticallyacceptable excipients and carriers are generally known to those skilledin the art and are thus included in the instant invention. Suchexcipients and carriers are described, for example, in “RemingtonsPharmaceutical Sciences” Mack Pub. Co., New Jersey (1991).

The formulations of the invention can be designed to be short-acting,fast-releasing, long-acting, and sustained-releasing. Thus, thepharmaceutical formulations can also be formulated for controlledrelease or for slow release.

Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose, i.e., control or thetreatment of disorders or diseases. More specifically, a therapeuticallyeffective amount means an amount of compound effective to prevent,alleviate or ameliorate symptoms/signs of disease or prolong thesurvival of the subject being treated.

The quantity of active component, which is the compound of thisinvention, in the pharmaceutical composition and unit dosage formthereof, may be varied or adjusted widely depending upon the manner ofadministration, the potency of the particular compound and the desiredconcentration. Determination of a therapeutically effective amount iswell within the capability of those skilled in the art. Generally, thequantity of active component will range between 0.01% to 99% by weightof the composition.

Generally, a therapeutically effective amount of dosage of activecomponent will be in the range of about 0.01 to about 100 mg/kg of bodyweight/day, preferably about 0.1 to about 10 mg/kg of body weight/day,more preferably about 0.3 to 3 mg/kg of body weight/day, even morepreferably about 0.3 to 1.5 mg/kg of body weight/day It is to beunderstood that the dosages may vary depending upon the requirements ofeach subject and the severity of the disorders or diseases beingtreated.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

Also, it is to be understood that the initial dosage administered may beincreased beyond the above upper level in order to rapidly achieve thedesired plasma concentration. On the other hand, the initial dosage maybe smaller than the optimum and the daily dosage may be progressivelyincreased during the course of treatment depending on the particularsituation. If desired, the daily dose may also be divided into multipledoses for administration, e.g., two to four times per day.

Medical and Veterinary Uses

Compounds of the present invention are Janus Kinase inhibitors (JAK-i)with efficacy against Janus Kinase-1 (JAK-1), Janus Kinase-2 (JAK-2) andJanus Kinase-3 (JAK-3). Accordingly, they are useful as therapeuticagents for organ transplants, lupus, multiple sclerosis, rheumatoidarthritis, psoriasis, Type I diabetes and complications from diabetes,cancer, asthma, atopic dermatitis, autoimmune thyroid disorders,ulcerative colitis, Crohn's disease, Alzheimer's disease, leukemia,osteoarthritis, control of pruritus, chronic respiratory disease andother indications where immunosuppression/immunomodulation would bedesirable.

In addition, there are substantial needs for safe and efficacious agentsto control atopic dermatitis in animals. The market for treating atopicdermatitis in animals is currently dominated by corticosteroids, whichcause distressing and undesirable side effects in animals, specificallyin companion animals such as dogs. Antihistamines are also used, but arepoorly effective. A canine formulation of cyclosporine (ATOPICA™) iscurrently being marketed for atopic dermatitis, but is expensive and hasa slow onset of efficacy. In addition, there are GI toleration issueswith ATOPICA™. Compounds of present invention are JAK inhibitors withefficacy against JAK-1 and JAK-3. These compounds will be an alternativeto steroid usage and provide resolution of chronic pruritus andinflammation that would either persist in atopic dermatitis or slowlyregress following removal of allergen or causative agent, such as fleasin flea-allergic dermatitis.

Compounds of the present invention may be administered in apharmaceutically acceptable form either alone or in combination with oneor more additional agents which modulate a mammalian immune system orwith antiinflammatory agents. These agents may include but are notlimited to cyclosporin A (e.g. Sandimmune® or Neoral®, rapamycin, FK-506(tacrolimus), leflunomide, deoxyspergualin, mycophenolate (e.g.Cellcept®, azathioprine (e.g. Imuran®), daclizumab (e.g. Zenapax®), OKT3(e.g. Orthocolone®), AtGam, aspirin, acctaminophen, ibuprofen, naproxen,piroxicam, and antiinflammatory steroids (e.g. prednisolone ordexamethasone). These agents may be administered as part of the same orseparate dosage forms, via the same or different routes ofadministration, and on the same or different administration schedulesaccording to standard pharmaceutical practice known to one skilled inthe art.

In one embodiment, the invention provides methods of treating orpreventing a disease, condition or disorder associated with JAK in asubject, such as a human or non-human mammal, comprising administeringan effective amount of one or more compounds described herein to thesubject. The JAK associated disease, condition or disorder can berelated to JAK-1, JAK-2, JAK-3, and/or Tyk-2. Suitable subjects that canbe treated include domestic or wild animals, companion animals, such asdogs, cats, horses and the like; livestock including, cows and otherruminants, pigs, poultry, rabbits and the like; primates, for examplemonkeys, such as rhesus monkeys and cynomolgus (also known ascrab-eating or long-tailed) monkeys, marmosets, tamarins, chimpanzees,macaques and the like; and rodents, such as rats, mice, gerbils, guineapigs and the like. In one embodiment, the compound is administered in apharmaceutically acceptable form, optionally in a pharmaceuticallyacceptable carrier.

JAK/STAT signaling has been implicated in the mediation of many abnormalimmune responses such as allergies, asthma, autoimmune diseases such astransplant (allograft) rejection, rheumatoid arthritis, amyotrophiclateral sclerosis and multiple sclerosis, as well as in solid andhematologic malignancies such as leukemia and lymphomas. For a review ofthe pharmaceutical intervention of the JAK/STAT pathway see Frank,(1999), Mol. Med. 5:432:456 and Seidel et al., (2000), Oncogene19:2645-2656.

JAK-3 in particular has been implicated in a variety of biologicalprocesses. For example, the proliferation and survival of murine mastcells induced by IL-4 and IL-9 have been shown to be dependent on JAK-3and gamma chain-signaling. Suzuki et al., (2000), Blood 96:2172-2180.JAK-3 also plays a crucial role in IgE receptor-mediated mast celldegranulation responses (Malaviya et al., (1999), Biochem. Biophys. Res.Commun. 257:807-813), and inhibition of JAK-3 kinase has been shown toprevent type I hypersensitivity reactions, including anaphylaxis(Malaviya et al., (1999), J. Biol. Chem. 274:27028-27038). JAK-3inhibition has also been shown to result in immune suppression forallograft rejection (Kirken, (2001), Transpl. Proc. 33:3268-3270). JAK-3kinases have also been implicated in the mechanism involved in early andlate stages of rheumatoid arthritis (Muller-Ladner et al., (2000), J.Immunal. 164:3894-3901); familial amyotrophic lateral sclerosis (Trieuet al., (2000), Biochem Biophys. Res. Commun. 267:22-25); leukemia(Sudbeck et al., (1999), Clin. Cancer Res. 5:1569-1582); mycosisfungoides, a form of T-cell lymphoma (Nielsen et al., (1997), Prac.Natl. Acad. Sci. USA 94:6764-6769); and abnormal cell growth (Yu et al.,(1997), J. Immunol. 159:5206-5210; Catlett-Falcone et al., (1999),Immunity 10:105-115).

The JAK kinases, including JAK-3, are abundantly expressed in primaryleukemic cells from children with acute lymphoblastic leukemia, the mostcommon form of childhood cancer, and studies have correlated STATactivation in certain cells with signals regulating apoptosis (Demoulinet al., (1996), Mol. Cell. Biol. 16:4710-6; Jurlander et al., (1997),Blood 89:4146-52; Kaneko et al., (1997), Clin. Exp. Immun. 109:185-193;and Nakamura et al., (1996), J. Biol. Chem. 271: 19483-8). They are alsoknown to be important to lymphocyte differentiation, function andsurvival. JAK-3 in particular plays an essential role in the function oflymphocytes, macrophages, and mast cells. Given the importance of thisJAK kinase, compounds which modulate the JAK pathway, including thoseselective for JAK-3, can be useful for treating diseases or conditionswhere the function of lymphocytes, macrophages, or mast cells isinvolved (Kudlacz et al., (2004) Am. J. Transplant 4:51-57; Changelian(2003) Science 302:875-878).

Conditions in which targeting of the JAK pathway or modulation of theJAK kinases, particularly JAK-3, are contemplated to be therapeuticallyuseful include, arthritis, asthma, autoimmune diseases, cancers ortumors, diabetes, certain eye diseases, disorders or conditions,inflammation, intestinal inflammations, allergies or conditions,neurodegenerative diseases, psoriasis, transplant rejection, and viralinfection. Conditions which can benefit for inhibition of JAK-3 arediscussed in greater detail below.

Accordingly, the compound of formula I or its pharmaceuticallyacceptable salts and pharmaceutical compositions can be used to treat avariety of conditions or diseases such as:

Arthritis, including rheumatoid arthritis, juvenile arthritis, andpsoriatic arthritis;

Asthma and other obstructive airways diseases, including chronic orinveterate asthma, late asthma, airway hyper-responsiveness, bronchitis,bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma,dust asthma, recurrent airway obstruction, and chronic obstructionpulmonary disease;

Autoimmune diseases or disorders, including those designated as singleorgan or single cell-type autoimmune disorders, for example Hashimoto'sthyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritisof pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis,Goodpasture's disease, autoimmune thrombocytopenia, sympatheticophthalmia, myasthenia gravis, Graves' disease, primary biliarycirrhosis, chronic aggressive hepatitis, ulcerative colitis andmembranous glomerulopathy, those designated as involving systemicautoimmune disorder, for example systemic lupus erythematosis,rheumatoid arthritis, Sjogren's syndrome, Reiter's syndrome,polymyositis-dermatomyositis, systemic sclerosis, polyarteritis nodosa,multiple sclerosis and bullous pemphigoid, and additional autoimmunediseases, which can be O-cell (humoral) based or T-cell based, includingCogan's syndrome, ankylosing spondylitis, Wegener's granulomatosis,autoimmune alopecia, Type I or juvenile onset diabetes, and thyroiditis;

Cancers or tumors, including alimentary/gastrointestinal tract cancer,colon cancer, liver cancer, skin cancer including mast cell tumor andsquamous cell carcinoma, breast and mammary cancer, ovarian cancer,prostate cancer, lymphoma, leukemia, including acute myelogenousleukemia and chronic myelogenous leukemia, kidney cancer, lung cancer,muscle cancer, bone cancer, bladder cancer, brain cancer, melanomaincluding oral and metastatic melanoma, Kaposi's sarcoma, myelomasincluding multiple myeloma, myeloproliferative disorders, proliferativediabetic retinopathy, and angiogenic-associated disorders includingsolid tumors;

Diabetes, including Type I diabetes and complications from diabetes;

Eye diseases, disorders or conditions including autoimmune diseases ofthe eye, keratoconjunctivitis, vernal conjunctivitis, uveitis includinguveitis associated with Behcet's disease and lens-induced uveitis,keratitis, herpetic keratitis, conical keratitis, corneal epithelialdystrophy, keratoleukoma, ocular premphigus, Mooren's ulcer, scleritis,Grave's ophthalmopathy, Vogt-Koyanagi-Harada syndrome,keratoconjunctivitis sicca (dry eye), phlyctenule, iridocyclitis,sarcoidosis, endocrine ophthalmopathy, sympathetic ophthalmitis,allergic conjunctivitis, and ocular neovascularization;

Intestinal inflammations, allergies or conditions including Crohn'sdisease and/or ulcerative colitis, inflammatory bowel disease, coeliacdiseases, proctitis, eosinophilic gastroenteritis, and mastocytosis;

Neurodegenerative diseases including motor neuron disease, Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis,Huntington's disease, cerebral ischemia, or neurodegenerative diseasecaused by traumatic injury, strike, glutamate neurotoxicity or hypoxia;ischemic/reperfusion injury in stroke, myocardial ischemica, renalischemia, heart attacks, cardiac hypertrophy, atherosclerosis andarteriosclerosis, organ hypoxia, and platelet aggregation;

Skin diseases, conditions or disorders including atopic dermatitis,eczema, psoriasis, scleroderma, pruritus and other pruritic conditions;

Allergic reactions including allergic dermatitis in mammal includinghorse allergic diseases such as bite hypersensitivity, summer eczema andsweet itch in horses.

Transplant rejection, including pancreas islet transplant rejection,bone marrow transplant rejection, graft-versus-host disease, organ andcell transplant rejection such as bone marrow, cartilage, cornea, heart,intervertebral disc, islet, kidney, limb, liver, lung, muscle, myoblast,nerve, pancreas, skin, small intestine, or trachea, and xenotransplantation; and

Another embodiment provides a method of inhibiting a JAK enzyme,including JAK-1, JAK-2, JAK-3 and/or Tyk-2, that includes contacting theJAK enzyme with either a non-therapeutic amount or a therapeuticallyeffective amount of one or more of the present compounds. Such methodscan occur in vivo or in vitro. In vitro contact can involve a screeningassay to determine the efficacy of the one or more compounds against aselected enzyme at various amounts or concentrations. In vivo contactwith a therapeutically effective amount of the one or more compounds caninvolve treatment of a described disease, disorder or condition orprophylaxis of organ transplant rejection in the animal in which thecontact occurs. The effect of the one or more compounds on the JAKenzyme and/or host animal can also be determined or measured. Methodsfor determining JAK activity include those described in the Examples aswell as those disclosed in WO 99/65908, WO 99/65909, WO 01/42246, WO02/00661, WO 02/096909, WO 2004/046112 or WO 2007/012953.

The following reaction schemes illustrate the general syntheticprocedures of the compounds of the present invention. All startingmaterials are prepared by procedures described in these schemes or byprocedures known to one of ordinary skill in the art.

It will be apparent to those skilled in the art that sensitivefunctional groups (Pg or Pg1) may need to be protected and deprotectedduring synthesis of a compound of the invention. This may be achieved byconventional methods, for example as described in “Protective Groups inOrganic Synthesis” by TW Greene and PGM Wuts, John Wiley & Sons Inc(1999), and references therein.

In Scheme I, 4-Chloro-7H-pyrrolo[2,3-d]pyrimidine (a) can be obtainedcommercially. trans-4-(Methylamino)-cyclohexyl]methanol (b) may beobtained from the corresponding carboxylic acid,trans-4-[(tert-butoxycarbonyl)amino]cyclohexanecarboxylic acid, upontreatment with a reducing agent such as lithium aluminium hydride in anaprotic, anhydrous solvent such as tetrahydrofuran at temperaturesbetween 0-60° C. for several hours.

As shown in Scheme I, a compound of structure (c) can be synthesized bythe reaction of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (a) withtrans-4-(methylamino)-cyclohexyl]methanol (b) in a suitable aprotic,polar solvent such as N,N-dimethylformamide, aqueous dioxane anddimethylsulfoxide, in the presence of a suitable base such astriethylamine and potassium carbonate at elevated temperatures up to 90°C. for up to a few hours.

A compound of structure (d) could be synthesized in a two step procedurefrom a compound of structure (c). For example, a compound of structure(d) would be synthesized by firstly using brominating reagents such asthionyl bromide or phosphorous tribromide in a polar, aprotic solventsuch as methylene chloride to afford the unprotectedcyclohexylmethylbromide, and secondly by addition of a suitableprotecting reagent such as tosyl chloride to give the protected compoundof structure (d).

A compound of structure (e) can be prepared by using simple protectionprocesses from a compound of structure (c). For example, when Pg and Pg₁are both tosyl, this can be effected in a one step reaction upontreatment of the unprotected compound of structure (c) with tosylchloride in the presence of a polar, aprotic solvent such as methylenechloride, a catalyst such as DMAP and a weak base such as triethylamine.

A compound of structure (f) may be synthesized from a compound ofstructure (e) by S-alkylation using a suitable nucleophile. Thuscompounds of structure (e) wherein the protecting group (Pg₁) is asuitable hydroxyl protecting group such as tosyl or mesyl may be reactedwith potassium thioacetate in a polar solvent such as dimethylsulfoxideor N-methylpyrrolidine at elevated temperatures up to 75° C. for up to 2hours to give compounds of structure (f).

A compound of structure (g) may be synthesized by an oxidation procedurefrom compounds of formula (f). Many oxidizing conditions are known tothose skilled in the art, for example those described in “Handbook ofReagents for Organic Synthesis—Oxidising and Reducing Agents” edited byS. D. Burke and R. L. Danheiser. For example, a compound of structure(f), optionally wetted with water, can be treated with formic acidfollowed by slow addition of hydrogen peroxide whilst stirring at roomtemperature for about 15 hours to give a compound of structure (g).Alternatively, oxone may be employed in a polar solvent such as aceticacid, if the reaction is performed in the presence of potassium acetatethe potassium salt of the compound of formula (g) is produced.

It is envisaged that a compound of structure (g) may be synthesizeddirectly from a compound of structure (e) upon treatment with a suitablesulfur nucleophile such as sodium sulfite in a polar solvent. Similarly,a compound of structure (g) could be synthesized from a compound ofstructure (d) upon nucleophilic substitution with sodium sulfite.

Treatment of sulphonic acids of formula (g) with a chlorinating agentsuch as thionyl chloride in an aprotic, polar solvent such as methylenechloride with a polar cosolvent such as N,N-dimethylformamide at refluxgives the chlorinated compounds. The chlorinated compound then reacts inan aprotic, anhydrous solvent such as tetrahydrofuran with suitableamines in neat, gaseous form, or dissolved in an aprotic, anhydroussolvents such as tetrahydrofuran, at room temperature to produce acompound of structure (h). Optionally an anhydrous, weak base such astriethylamine may be used to mop up hydrochloric acid generated in thereaction.

Compounds of formula I of the present invention may be prepared fromcompounds of formula (h) wherein Pg is a suitable protecting group bydeprotection procedures known to one skilled in the art. For example,when the protecting group (Pg) is tosyl, suitable deprotectionconditions involve reaction with a base such as lithium hydroxide orpotassium hydroxide in a protic solvent such as methanol or isopropanoland optionally miscible cosolvents such as tetrahydrofuran and water atroom temperature for several hours, to produce the deprotected amine offormula I.

Salts of compounds of formula I may be formed by the reaction of thefree base of compounds of formula I with a suitable acid such as maleicacid in the presence of a protic solvent such as butanol and optionallya cosolvent such as water.

Alternatively, compounds of this invention can be prepared in accordancewith Scheme II.

In Scheme II,4-methyl-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidine (j)can be obtained from commercially available (a) using a protecting agentsuch as tosyl chloride using procedures well known in the art.trans-4-(Methylamino)-cyclohexyl]methanol (b) may be obtained from thecorresponding carboxylic acid,trans-4-[(tert-butoxycarbonyl)amino]cyclohexanecarboxylic acid, upontreatment with a reducing agent such as Vitride in an anhydrous solventsuch as toluene at temperatures between 0-110° C. for several hours.

As shown in Scheme II, a compound of structure (k) can be synthesized bythe reaction of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (j) withtrans-4-(methylamino)-cyclohexyl]methanol (b) in a suitable solvent suchas acetone, in the presence of a suitable base such as triethylaminewith a catalytic amount of potassium iodide at elevated temperatures upto 60° C. for up to a few hours.

A compound of structure (k) could synthesized by addition of a suitablemesylating reagent such as mesyl chloride in the presence of a suitablebase such as triethylamine or diethylisopropylamine in a suitablesolvent such as acetone at elevated temperatures up to 60° C. to givethe methane sulfonyl compound of structure (k).

A compound of structure (l) can be prepared by using simple S-alkylationprocesses from a compound of structure (k) using a suitable nucleophile.Thus compounds of structure (k) may be reacted with sodium sulphite insolvent such as isopropylalcohol or water or toluene at elevatedtemperatures up to 90° C. for up to 4 hours to give compounds ofstructure (l).

Treatment of sulphonic acids of formula (l) with a chlorinating agentsuch as thionyl chloride in an aprotic, polar solvent such as THF ormethylene chloride with a polar cosolvent such as N,N-dimethylformamideat temperatures between 0 and 40° C. gives the chlorinated compounds.The chlorinated compound then reacts in an aprotic, anhydrous solventsuch as tetrahydrofuran with suitable amines such as methyl amine,cyclobutyl amine or 2-hydroxyazetidine preferably in neat, gaseous form,or dissolved in an aprotic, anhydrous solvents such as tetrahydrofuran,at room temperature to produce a compound of structure (h). Optionallyan anhydrous, weak base such as triethylamine may be used to mop uphydrochloric acid generated in the reaction.

EXAMPLES Preparation 1N-Methyl-1-[trans-4-(methyl{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclohexyl]methanesulfonamide

Method (a) To a solution of the compound of Preparation 2 (308.0 g wetweight, 214.5 g dry weight, 0.41 mol) in tetrahydrofuran (1.0 l) isadded methylamine (2M in tetrahydrofuran, 687 ml) over 1 h. Afterstirring at room temperature for 30 min, additional methylamine (2M intetrahydrofuran, 53 ml) is added and the reaction mixture is stirred atroom temperature for 18 h. The mixture is reduced in volume to 600 ml,via vacuum distillation, and tetrahydrofuran (300 ml) is added, beforethe mixture is again reduced in volume to approximately 750 ml. To themixture, heated at 45° C., is added 2-propanol (247 ml) and water (693ml). After cooling to room temperature, the solid material is collectedby filtration, washed with water (2×250 ml) and dried in vacuo at 65° C.to give the title compound (180.4 g).

¹H-NMR (d₆-DMSO): 1.17-1.32 (2H), 1.57-1.73 (4H), 1.76-1.92 (1H),1.93-2.08 (2H), 2.30-2.39 (3H), 2.53-2.62 (3H), 2.87-2.98 (2H),3.07-3.17 (3H), 4.53-4.75 (1H), 6.81-6.94 (1H), 7.38-7.47 (2H),7.56-7.65 (1H), 7.92-8.02 (2H), 8.15-8.27 (1H).

Method (b) To a solution of the compound of Preparation 2 (165 g, 0.34mol) in THF (1.65 l) and N,N-dimethylformamide (5.0 ml) at 0-5° C. isadded thionyl chloride (125 ml, 17 mol), over 25 min. The reactionmixture was stirred for 30 minutes at 0-5° C. then slowly heated to 40°C. for 8 h. After cooling to r.t. the solvent was evaporated underreduced pressure and azeotroped with THF to removed thionyl chloride. Tothe sulphonyl chloride obtained was added fresh THF (1.65 l) and themixture cooled to 0° C. Dry N-methylamine gas was purged for 30 minutesand the reaction stirred for a further 4 hours at r.t. The solvent wasevaporated to half of its volume (800 ml) and heptane (1.5 l) added. Theproduct precipitated out and was filtered and washed with water (1 l) togive the title compound (80 g).

¹H-NMR (d₆-DMSO): 1.17-1.32 (2H), 1.57-1.73 (4H), 1.76-1.92 (1H),1.93-2.08 (2H), 2.30-2.39 (3H), 2.53-2.62 (3H), 2.87-2.98 (2H),3.07-3.17 (3H), 4.53-4.75 (1H), 6.81-6.94 (1H), 7.38-7.47 (2H),7.56-7.65 (1H), 7.92-8.02 (2H), 8.15-8.27 (1H)

Preparation 2[trans-4-(Methyl{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclohexyl]methanesulfonylchloride

To a solution of the compound of Preparation 3 (210.0 g, 0.42 mol) indichloromethane (1.2 l) and N,N-dimethylformamide (4.1 ml) is addedthionyl chloride (151.0 ml, 2.1 mol), over 25 min. The reaction mixtureis heated at reflux for 18 h and then reduced in volume to 800 ml, viavacuum distillation. To the mixture, heated at approximately 30° C., isadded ethyl acetate (1.1 l) over 1 h, followed by heptane (546 ml),added over 20 min at room temperature. The mixture is cooled to 0° C.and stirred for 1 h and the resulting precipitate is collected byfiltration under nitrogen. The solid is washed with heptane (2×125 ml)to give the title compound (308.0 g wet weight), which is stored undernitrogen and used directly.

Preparation 3[trans-4-(Methyl{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclohexyl]methanesulfonicacid

Method (a) To a mixture of the compound of Preparation 4 (100.0 g wetweight, 23.5 g dry weight, 47.8 mmol) and formic acid (82.0 g, 68.0 ml,1.8 mol) is added hydrogen peroxide (35 wt. % in water, 21.0 ml, 0.26mol) over 10 min. The reaction mixture is stirred at room temperaturefor 15 h and then quenched by addition to an aqueous sodiummetabisulfate or metabisulfite solution (33 wt. %, 35 ml). To themixture is added water (5 ml), 2-propanol (50 ml) and aqueous sodiumhydroxide solution (33 wt. %, 161 ml) and the slurry is stirred at roomtemperature for 1 h. The solid material is collected by filtration,washed with water (100 ml) and dried in vacuo at 60° C. to give thetitle compound (26.0 g).

¹H-NMR (d₆-DMSO): 0.98-1.18 (2H), 1.55-1.76 (5H), 1.99-2.13 (2H),2.29-2.39 (5H), 3.05-3.15 (3H), 4.47-4.76 (1H), 6.77-6.92 (1H),7.38-7.48 (2H), 7.54-7.62 (1H), 7.91-8.02 (2H), 8.17-8.25 (1H)

Method (b) To a solution of the compound of Preparation 3 in IPA-water(585 ml each, 1:1, V/V) was added sodium sulphate and the mixture washeated to 80-90° C. for 24 hours. After allowing to cool to r.t. thesolvent was evaporated up to 50% and the pH of the reaction mixtureadjusted in the range 3-4 by addition of acetic acid. Toluene (1 l) wasadded and the mixture evaporated to 80%. Further toluene (1 l) was addedand the mixture refluxed for 4 hours. The toluene was decanted and theresultant title compound obtained by drying under vacuum (168 g).

¹H-NMR (d₆-DMSO): 0.98-1.18 (2H), 1.55-1.76 (5H), 1.99-2.13 (2H),2.29-2.39 (5H), 3.05-3.15 (3H), 4.47-4.76 (1H), 6.77-6.92 (1H),7.38-7.48 (2H), 7.54-7.62 (1H), 7.91-8.02 (2H), 8.17-8.25 (1H)

Preparation 4S-{[trans-4-(Methyl{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclohexyl]methyl}ethanethioate

To a solution of potassium thioacetate (11.4 g, 99.4 mmol) in dimethylsulfoxide (30 ml) is added the compound of Preparation 5 (50.0 g, 87.9mmol) in dimethyl sulfoxide (130 ml). The reaction mixture is heated at55° C. for 3 h, cooled to room temperature and quenched by addition toan aqueous sodium hydrogen carbonate solution (0.1M, 640 ml). Themixture is cooled to 13° C. and the resulting precipitate is collectedby filtration and washed with water (250 ml) to give the title compound(204.0 g wet weight).

¹H-NMR (d₆-DMSO): 1.06-1.23 (2H), 1.39-1.51 (1H), 1.51-1.70 (4H),1.74-1.88 (2H), 2.30-2.40 (6H), 2.73-2.84 (2H), 3.06-3.14 (3H),4.44-4.76 (1H), 6.76-6.94 (1H), 7.36-7.49 (2H), 7.56-7.62 (1H),7.90-8.02 (2H), 8.17-8.26 (1H)

Preparation 5[trans-4-(Methyl{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclohexyl]methyl4-methylbenzenesulfonate

To a solution of the compound of Preparation 6 (42.0 g, 0.16 mol) indichloromethane (1 l) is added triethylamine (68.3 g, 0.68 mol) and4-dimethylaminopyridine (1.0 g, 8.2 mmol), followed by p-toluenesulfonylchloride (62 g, 0.33 mol). The reaction mixture is stirred at roomtemperature for 2 h, before addition of further p-toluenesulfonylchloride (45.5 g, 0.24 mol). After stirring for 18 h, the mixture isconcentrated in vacuo and a portion of the residue (approximately 309 g)is slurried in methanol (758 ml) for 15 min. To the slurry is addedwater (600 ml) and saturated aqueous sodium hydrogen carbonate solution(142 ml) and the mixture is stirred for 1 h. The solid material iscollected by filtration and washed with methanol:water [1:1, 50 ml],water (50 ml) and hexanes (50 ml). The solid is dried in vacuo at 60° C.to give the title compound (87.1 g).

¹H-NMR (d₆-DMSO): 1.02-1.20 (2H), 1.53-1.75 (7H), 2.31-2.39 (3H),2.39-2.47 (3H), 3.04-3.12 (3H), 3.80-3.91 (2H), 4.34-4.76 (1H),6.78-6.93 (1H), 7.37-7.54 (4H), 7.54-7.64 (1H), 7.75-7.84 (2H),7.91-8.01 (2H), 8.14-8.25 (1H)

Preparation 6{trans-4-[Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}-methanol

A mixture of the compound of trans-4-(Methylamino)cyclohexyl]methanol(may be prepared according to the procedure described in WO2002/14267)(50.0 g, 0.35 mol), 4-chloro-7H-pyrrolo[2,3-d]pyrimidine(commercially available, 42.9 g, 0.27 mol) and potassium carbonate (57.3g, 0.42 mol) in water (1 l) and 1,4-dioxane (100 ml) is heated at 90° C.for 15 h. To the mixture is added the compound of Preparation 7 (2.0 g,14.0 mmol) and the reaction mixture is heated at 90° C. for a further 1h. After cooling to room temperature, the mixture is stirred for 1 h andthe solid material is collected by filtration, washed with water (150ml) and dried in vacuo at 65° C. to give the title compound (72.7 g).

¹H-NMR (d₆-DMSO): 1.00-1.19 (2H), 1.30-1.45 (1H), 1.52-1.77 (4H),1.77-1.91 (2H), 3.09-3.20 (3H), 3.20-3.29 (2H), 4.37-4.51 (1H),6.45-6.57 (1H), 7.06-7.17 (1H), 8.01-8.14 (1H)

Preparation 7[trans-4-(Methyl{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclohexyl]methanesulfinate

To a solution of the compound of Preparation 4 (60.0 g, 0.418 mol) inacetone (600 ml) is added triethylamine (117.5 ml, 0.837 mol) andcatalytic potassium iodide (3.4 g, 0.05 mol), followed by4-chloro-7H-pyrrolo[2,3-d]pyrimidine (commercially available, 102.8 g,0.335 mol). The resulting mixture was heated to 60° C. for 22 h. Afterallowing to cool to r.t. acetone (300 ml) was added, followed bytriethylamine (146.9 ml, 1.04 mol) then mesyl chloride (81.7 ml, 1.047mol). After stirring for 4 hours at r.t. water was added (1.8 l)whereupon the product precipitated out. The product was filtered, driedand triturated with a mixture of MTBE-heptane (6:4, 600 ml). A secondtrituration from MTBE-heptane was conducted and the resultant titlecompound obtained (120 g).

¹H-NMR (d₆-DMSO):

Preparation 8 [trans-4-(Methylamino)cyclohexyl]methanol

Vitride solution (65%, 767 ml, 2.465 mol) was added dropwise over 1 h toa solution of trans-4-[(tert-butoxycarbonyl)amino]cyclohexanecarboxylicacid (commercially available, 100 g, 0.4109 mol) in toluene (1 l). Afterthe addition was complete the reaction mixture was heated to reflux for100-110° C. The reaction mixture was quenched with aq. Sodium sulphatesolution (800 ml) at temperatures below 10° C. The reaction mixture wasfiltered through celite and the filter cake washed with DCM (500 ml)followed by water (100 ml). The organics layer was separated and theaqueous layer extracted twice with DCM (600 ml then 400 ml). Thecombined organics layers were dried over sodium sulphate andconcentrated in vacuo to give the title compound (62 g).

¹H-NMR (CD₃OD): 1.08-1.31 (4H), 1.51-1.64 (1H), 1.93-2.05 (2H),2.10-2.22 (2H), 2.38-2.50 (1H), 2.50-2.54 (3H), 3.48-3.55 (2H)

Example 1a Preparation ofN-methyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}methanesulfonamide

To a solution of the compound of Preparation 1 (250.0 g, 0.48 mol) in2-propanol (1.2 l) is added lithium hydroxide (48.7 g, 2.03 mol) inwater (1.2 l). The reaction mixture is heated at 40° C. for 8 h and thenstirred at room temperature for 18 h. The mixture is filtered, washingthrough with 2-propanol:water (1:1, 100 ml) and the filtrate is adjustedto pH 7.5 by addition of hydrochloric acid (6N). After stirring for 1 h,the solid material is collected by filtration, washed with2-propanol:water (1:2, 240 ml) and dried in vacuo at 60° C. to give thetitle compound (148.7 g) as a free base (Example 1a).

¹H-NMR (d₆-DMSO): 1.20-1.39 (2H), 1.62-1.75 (4H), 1.77-1.91 (1H),1.97-2.11 (2H), 2.54-2.63 (3H), 2.89-2.99 (2H), 3.10-3.21 (3H),4.44-4.86 (1H), 6.43-6.61 (1H), 7.01-7.19 (1H), 7.94-8.16 (1H)

Example 1b Preparation ofN-methyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}methanesulfonamidemaleic acid salt

A mixture of the compound of Example 1a (212.0 g, 628.3 mmol) and maleicacid (67.2 g, 579.0 mmol) in 1-butanol (3200 ml) and water (400 ml) isstirred at room temperature for 18 h. The mixture is reduced in volumeto 1600 ml, via vacuum distillation (55° C., 100 mbar) and then cooledto 0° C. The resulting solid is collected by filtration, washed withheptane (500 ml) and dried in vacuo at 35° C. to give the maleate saltofN-methyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-cyclohexyl}methanesulfonamide(253.0 g) as a crystalline form known as Form A.

Experimental MH⁺ 338.2; expected 338.2.

¹H-NMR (d₆-DMSO): 1.24-1.38 (2H), 1.68-1.92 (5H), 2.00-2.11 (2H),2.56-2.61 (3H), 2.91-3.00 (2H), 3.15-3.27 (3H), 4.39-4.70 (1H),6.53-6.73 (1H), 7.16-7.36 (1H), 8.07-8.29 (1H).

Example 1c Method for Collecting Powder X-ray Diffraction forN-methyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}-methanesulfonamideMaleic Acid Salt (Form A)

The powder X-ray diffraction patterns for Form A,N-methyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}-methanesulfonamidemaleic acid salt were collected using a Bruker-AXS Ltd. D4 powder X-raydiffractometer fitted with an automatic sample changer, a theta-thetagoniometer, automatic beam divergence slit, and a PSD Vantec-1 detector.The sample was prepared for analysis by mounting on a low backgroundcavity silicon wafer specimen mount. The specimen was rotated whilstbeing irradiated with copper K-alpha₁ X-rays (wavelength=1.5406Ångstroms) with the X-ray tube operated at 40 kV/35 mA. The analyseswere performed with the goniometer running in continuous mode set for a0.2 second count per 0.018° step over a two theta range of 2° to 55°.The results are summarized in Table 1 and Table 2.

TABLE 1 Powder X-ray Diffraction Peaks expressed in degrees 2-theta,±0.2 degrees, approximately Angle Intensity Angle Intensity AngleIntensity 2-theta I % 2-theta I % 2-theta I % 6.178 72.6 17.997 32.324.86 19.8 8.519 24.5 18.539 44.5 25.602 10.7 12.601 88.4 20.298 18.226.582 13 13.819 38 20.659 27.8 27.02 30.6 15.478 34.3 21.583 11.127.721 18.7 15.719 100 22.642 12.7 28.161 23.7 16.32 27.1 23.08 12.128.38 28.9

TABLE 2 Selected Powder X-ray Diffraction Peaks expressed in degrees2-theta, ±0.2 degrees, approximately Angle Lattice Dimension Intensity2-theta d(A) I % 6.179 14.3 73 12.601 7.01 88 15.719 5.63 100 18.5394.78 45 27.02 3.29 30.6 28.38 3.14 28.9

As is readily apparent to one skilled in the art, the results of anyX-ray powder diffraction may vary and subsequent XRPD's will not beidentical, even when carried out on the same lot of material. Thisvariance can be due to test sample preparation, temperature, theparticular model of X-ray diffractometer used, the operator's technique,etc. The term “approximately” if used in defining a peak in an X-raypowder diffraction pattern is defined as the stated 2θ value±0.2°2θ. Anydetermination of whether a crystalline form is the Form A polymorph andencompassed by the claims should be interpreted in light of thevariability in this test.

This variability is demonstrated in FIG. 1. The two different lots ofN-methyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}-methanesulfonamidemaleic acid salt Form A were submitted to the same XRPD diffractometer.The characteristic peaks in FIG. 1 confirming that it is the Form Apolymorph. However, the relative intensity of these peaks as well as theother identifying peaks varied slightly.

Example 2 Preparation ofN-cyclobutyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}methanesulfonamide

Following the general procedure of Example 1 and making non-criticalvariations but substituting the precursor to cyclobutanamine, the titlecompound is provided.

Experimental MH⁺ 378.0; expected 378.2

¹H-NMR (d₆-DMSO): 1.22-1.32 (2H), 1.47-1.70 (6H), 1.78-2.04 (5H),2.16-2.24 (2H), 2.85-2.86 (2H), 3.15 (3H). 3.68-3.78 (1H), 4.60-4.72(1H), 6.51-6.54 (1H), 7.11-7.12 (1H), 7.44-7.49 (1H), 8.08 (1H), 11.60(1H)

Example 3 Preparation ofN-ethyl-1-{trans-4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl}methanesulfonamide

Following the general procedure of Example 1 and making non-criticalvariations but substituting the precursor to ethanamine, the titlecompound is provided.

Experimental MH⁺ 352.0; expected 352.2

¹H-NMR (CDCl₃): 1.24-1.44 (5H), 1.64-1.74 (2H), 1.87-2.09 (3H),2.15-2.21 (2H), 2.97-2.99 (2H), 3.18-3.27 (5H), 4.46-4.52 (1H),4.74-4.86 (1H), 6.55 (1H), 7.04 (1H), 8.28 (1H)

Example 4 JAK Enzymatic Assay

Materials: Recombinant JAK-2 (Catalog Number PV4210) and JAK-3 (CatalogNumber PV3855) were purchased from (Invitrogen Corporation, Madison,Wis.). Recombinant JAK-1 (GST-JAK-1 (852-1142)) and Tyk-2 (GST-Tyk2(870-1187, C1187S)) used in this study were expressed and purified atPfizer Laboratories. Adenosine 5′-triphosphate (ATP) was obtained fromSigma Chemical Company, St. Louis, Mo. The JAKtide peptide (peptidesequence, FITC-KGGEEEEYFELVKK (SEQ ID NO:1)) used for the JAK-2 andJAK-3 assays and the IRS-1 peptide (peptide sequence,5-FAM-KKSRGDYMTMQIG (SEQ ID NO:2)) used for the JAK-1 and Tyk-2 assayswere purchased from (American Peptide Company, Sunnyvale, Calif.).Coating Reagent 3 was purchased from (Caliper Life Sciences, Hopkinton,Mass.).

Methods: A peptide mobility shift assay was used to quantify thephosphorylation of the JAKtide (JAK-2 and JAK-3) or the IRS-1 peptide(JAK-1 and Tyk-2). Reactions were carried out in a 384-well plate(Matrical MP-101) in a 10 microliter total volume. Reaction mixturescontained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovineserum albumin (BSA), 0.0005% Tween-20, ATP (4 micromolar for JAK-2 andJAK-3, 40 micromolar for JAK-1 and 7 micromolar for Tyk-2)), 2% DMSO and1 micromolar peptide substrate (JAKtide for JAK-2 and JAK-3 or IRS-1peptide for JAK-1 and Tyk-2). Compounds were diluted serially in 100%dimethyl sulfoxide and tested in an 11 point dose response in duplicateor quadruplicate (200 nl of compound/DMSO was added per 10 microliterreaction). The reactions were initiated by the addition of enzyme to thefinal concentration of 2 nM JAK-2, 1 nM JAK-3, 7 nM Tyk2 or 20 nM JAK-1.The assay was run for 240 minutes for JAK-1, 150 minutes for JAK-2, 90minutes for JAK-3 and 60 minutes for Tyk-2. The assays were stopped atthe specified times with 20 microliter of 140 mM HEPES, 22.5 mM EDTA and0.15% Coating Reagent 3. The plates were placed on a LabChip 3000(LC3000) instrument (Caliper Life Sciences) to measure the formation ofphosphorylated peptide. Data was analyzed using Hits Well AnalyzerSoftware from (Caliper Life Sciences) to obtain the amount of productformed.

Data was then imported into an internal application where each datapoint was expressed as % inhibition based on uninhibited and no enzymecontrols. Dose-response data was then fit using a 4 parameter logisticequation (Equation 1) to determine an IC₅₀ value.

$\begin{matrix}{y = {\frac{\max - \min}{1 + \left( \frac{x}{{IC}_{50}} \right)^{s}} + \min}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Where max is the fitted uninhibited value, min is the fitted completeinhibition value, and s is the slope factor.

Using this protocol, the following results were generated for the titlecompounds of Example 1 and 2.

TABLE 3 JAK Enzymatic Assay Result Ex. # JAK-1 IC₅₀ JAK-2 IC₅₀ JAK-3IC₅₀ Tyk-2 IC₅₀ 1a 9.53 nM  17.5 nM 95.1 nM 75.1 nM 2 45.0 nM 101.0 nM 742 nM —

Example 5 Canine in vitro T-cell Proliferation Assay

T-cell activation plays a key role in a variety of inflammatory andautoimmune disorders as well as asthma, allergies and pruritus. SinceT-cell activation can, in part, can be triggered by cytokines thatsignal through the JAK-STAT pathway, a JAK inhibitor could be effectiveagainst such diseases involving aberrant T-cell activation.

Methods: Canine whole blood was collected in sodium heparin tubes from29 beagle dogs and 23 mixed breed dogs. Whole blood (20 μL) was platedin 96-well plates (Costar 3598) with 180 μL of medium (RPMI 1640, Gibco#21870-076, with 1% heat inactivated fetal bovine serum, Gibco#10082-39, 292 μg/ml L-glutamine, Gibco #250030-081, 100 u/ml penicillinand 100 μg streptomycin per ml, Gibco #15140-122) containing vehiclecontrol or test compound (0.001 to 10 μM), concanavalin A (ConA; 1μg/ml, Sigma C5275), and canine interleukin-2 (IL-2; 50 ng/ml, R&DSystems 1815-CL/CF). Wells containing whole blood, medium with vehiclecontrol and no ConA or IL-2 were used as background controls. Plateswere incubated at 37° C. for 48 hours. Tritiated thymidine, 0.4 μCi/well(Perkin Elmer, Net027A-005MC), was added for 20 additional hours. Plateswere frozen and then thawed, washed and filtered using a Brandel MLR-96cell harvester and prewet filter mats (Wallac 1205-401, Perkin Elmer).Filters were dried at 60° C. for one hour (Precision 16EG convectionoven) and placed into filter sample bags (Wallac 1205-411, Perkin Elmer)with 10 mL of scintillant (Wallac 1205-440, Perkin Elmer). Sealedfilters were counted on a LKB Wallac 1205 Betaplate liquid scintillationcounter. Data were collected via Gterm Betaplate program v1.1 (Wallaccopyright 1989-1990) and transformed into percent inhibition, calculatedusing the following formula:

${100 - \left\lbrack {\frac{\left( {{{Mean}\mspace{14mu}{Drug}\mspace{14mu}{Treatment}\mspace{14mu}{cpm}} - {{Mean}\mspace{14mu}{BCK}\mspace{14mu}{cpm}}} \right)}{\left( {{{Mean}{\mspace{11mu}\;}{Non}\text{-}{Drug}\mspace{14mu}{Treatment}\mspace{14mu}{cpm}} - {{Mean}\mspace{14mu}{BCK}\mspace{14mu}{cpm}}} \right)} \times 100} \right\rbrack} = {\%\mspace{14mu}{Inhibition}}$Data were graphically displayed as percent inhibition using GraphPadPrism 4.0, and IC₅₀ curves were fitted using a point to point analysis.

Results The average IC₅₀ values obtained when using whole blood frombeagles was 66.3 nM for the compound of Example 1a; 410 nM for Example2; and 83 nM for Example 3. The average IC₅₀ value obtained when usingwhole blood from mixed breed dogs was 138 nM for the compound of Example1a. These data suggest that the compounds of the present invention areeffective in inhibiting T-cell proliferation, a key feature in manydiseases.

Example 6 Reducing Flea-associated Pruritus and Dermatitis Assay

Flea-associated pruritus and dermatitis are common skin conditions indogs. Pruritus is one of the most severe clinical signs associated withflea associated dermatitis, and continued scratching, face rubbing, andfoot chewing can lead to a variety of changes to the skin such aserythema, edema, alopecia, lichenification, and hyper pigmentation.Flea-associated pruritus and dermatitis can be induced experimentally.In these models, inflammatory cells and cytokines have been shown tomediate immune reactions to allergens. Therefore, a JAK inhibitor thatinhibits signal transduction of pruritogenic and proinflammatorycytokine receptors could be effective in inhibiting, reducing orminimizing flea-associated pruritus and dermatitis.

Study Design

Twenty eight male and female dogs of mixed breeds ranging in weight from5-35 kg and greater than one year of age were infested withapproximately 100 unfed adult cat fleas (Ctenocephalides felis) 14 daysprior to the start of dosing and reinfested with 30 fleas per dog every4 days throughout the study. Seven days before dosing, twenty four dogswere randomized into three different treatment groups, placebo, 0.5mg/kg or 0.25 mg/kg of the compound of Example 1b, based on visualanalog scale (VAS) scores for skin lesions. Treatments were given orallytwice a day for 28 days, and pruritic behavior as well as erythema andskin lesions were was assessed during the study. Pruritic behavior wasrecorded by placing dogs into pens with video recording capability, andrecording their activity over 4 hours. Pruritic activity was quantitatedby determining how many seconds the dogs spent scratching. Skin lesionswere recorded by image capture of abdominal, inguinal region and theseverity ranked according to a visual analog scale (VAS).

Statistical Analysis

Video captured elapsed time of pruritic behavior was analyzed using amixed linear model for repeated measures. The model included fixedeffects of treatment and day of study and the interaction of treatmentand day of study. Random effects included block, the interaction ofblock and treatment and error. Baseline data for pruritic behavior(day-1) were used as covariate in the analysis of pruritic behavior.Least squares means were used as estimates of treatment means. Standarderrors of least squares means were estimated and 90% confidenceintervals were constructed. Geometric means were computed from the leastsquares means for log-transformed data. A priori contrasts were used toassess treatment. Treatment differences were assessed at the 10% levelof significance (P≦0.10).

Results

The treatment results are shown in FIG. 2 and FIG. 3. Lesions anderythema were significantly reduced in the 0.5 mg/kg group. FIG. 2illustrates Day 27 VAS Scores for Example 1b in flea allergic dogs(Least Squares Means). Significant reductions in pruritus as compared toplacebo at a 10% level of significance were seen at various time pointsduring the study for both groups (days 1, 4 and 12 for the 0.25 mg/kgdose and on days 1 and 20 for the 0.5 mg/kg dose). FIG. 3 illustratesSeconds of Pruritus per 4 hour recording for example 1b in flea allergicdogs (Long Geometric Mean).

Example 7 Cell Proliferation Inhibitory Assay

Feline Cell Lines The MYA-1 and FETJ are feline T-lymphoblast cell linesobtained from ATCC (Manassas, Va.). These cells were cultured in RPMI1640 complete media supplemented with 10% FBS at 37° C. in a humidifiedincubator with 5% CO₂.

Ex vivo Canine Lymphoma Nodal Tissue

Malignant lymph nodes were excised by veterinary staff at Michigan StateUniversity (MSU) Veterinary College, placed into transport media(Advanced RPMI 1640 complete medium supplemented with 10% Fetal BovineSerum (FBS), 100 U/mL penicillin, 100 ug/mL streptomycin and 0.25 ug/mLAmphotercin B (Invitrogen/Gibco®). Nodes were processed within 24 hoursof removal by mincing into tiny pieces and passing through a tissuesieve. Cell suspensions were spun at 200×g, supernatant was removed, andthe cell pellet was resuspended in NH₄Cl for 10 minutes at roomtemperature. The cell suspension was pelleted by centrifugation; theNH₄Cl was removed and washed once with Hanks Balanced Salt Solution(HBSS), followed by re-suspension in Proliferation Medium (Advanced RPMIcomplete, 1% FBS, 50 nM 2-Mercaptoethanol, 100 U/mL penicillin, 100ug/mL streptomycin and 0.25 ug/mL Amphotercin B). The cell suspensionwas then passed through a 100 μm nylon cell strainer (BD-Falcon) andcounted using a hemacytometer. Cells were cultured in eitherProliferation Medium alone, Proliferation Medium supplemented with0.005% Pansorbin® (Heat inactivated, formalin-fixed StaphylococcusAureus cells (SAC), Calbiochem), and 10 ng/mL canine IL-2 (R&D Systems),or Proliferation Medium supplemented with 125 ng/mL concavalin A (Sigma)and 125 ng/mL lipopolysaccarride (LPS; Calbiochem).

In vitro Anti-Proliferation Assay Method

Cells cultured in medium described above were plated in 96-well Costarplates (Corning) at a density of 1×10³ cells/well (feline cell lines) or2×10⁵ cells/well (lymph node cells) and exposed to variousconcentrations of test compounds for up to 5 days at 37° C. in ahumidified incubator with 5% CO₂. Effects on proliferation weredetermined using the CellTiter 96® AQ_(ueous) Non-Radioactive CellProliferation Assay (Promega) according to manufacturer's instructions.In general, proliferation was indirectly measured using a solubletetrazolium salt (MTS) and an electron coupling agent. MTS bioreductioninto a formazan product soluble in tissue culture medium was monitoredby absorbance at 490 nM on a Spectramax plate reader using Softmax Pro4.6 software (Molecular Devices). Data were graphically displayed aspercent DMSO control using GraphPad Prism 4.00, and IC₅₀ curves werefitted using a non-linear regression model with a sigmoidal doseresponse.

Results

Table 4 demonstrates that the compound of Example 1 can inhibitproliferation of the feline lymphoid cell line MYA-1 that is dependenton IL-2 for proliferation, but not an IL-2 independent line (FETJ). Thecompound of formula IA or its salt can also inhibit proliferation ofcanine nodal tissue obtained from dogs diagnoses with T- or B-celllymphoma. These results suggest a JAK inhibitor may be effective intreating canine and feline lymphomas.

TABLE 4 Cell Line or Stimulant in Species Lymph Node Description CultureMedium IC50 (nM) Feline MYA-1 Lymphoid line 122 (n = 2) Feline FETjLymphoid line >1000 Canine MSU LN 8 De novo T-cell LPS + ConA 357lymphoma Canine MSU LN 8 De novo T-cell SAC + IL-2 38 lymphoma CanineMSU LN 9 De novo B-cell LPS + ConA 147 lymphoma Canine MSU LN 9 De novoB-cell SAC + IL-2 100 lymphoma Canine MSU LN 10 Chemotherapy LPS + ConA687 resistant B-cell lymphoma- Canine MSU LN 11 De novo B-cell LPS +ConA 64 lymphoma

What is claimed is:
 1. A method for treating an autoimmune disease ordisorder selected from autoimmune thrombocytopenia, autoimmune hemolyticanemia, systemic lupus erythematosus, and bullous pemphigoid in a mammalcomprising administering to a mammal in need thereof a therapeuticallyeffective amount of a compound of formula I:

or a pharmaceutically acceptable salt thereof wherein R¹ is C₁₋₄alkyloptionally substituted with hydroxy.
 2. The method of claim 1 whereinthe therapeutically effective amount is from 0.01 mg/kg of bodyweight/day to 100 mg/kg of body weight/day.
 3. The method of claim 1wherein the therapeutically effective amount is from 0.1 mg/kg of bodyweight/day to 10 mg/kg of body weight/day.
 4. The method of claim 1wherein the mammal comprises companion animals.
 5. The method of claim 4wherein the companion animals are dogs, cats, and horses.
 6. The methodof claim 1 wherein the compound of formula I is administered orally,parenterally, or topically.
 7. A method for treating cancer selectedfrom mammary cancer, bone cancer, prostate cancer, bladder cancer, andmelanoma in a mammal comprising administering to a mammal in needthereof a therapeutically effective amount of a compound of formula I ofclaim
 1. 8. A method for treating keratoconjunctivitis andkeratoconjunctivitis sicca in a mammal comprising administering to amammal in need thereof a therapeutically effective amount of a compoundof formula I of claim
 6. 9. The method of claim 1 wherein R¹ of formulaI is methyl.